The present invention relates to enzymes, to polynucleotides encoding enzymes and to uses of enzymes and polynucleotides.
Protein kinase B (PKB) [1] or RAC protein kinase [2] is the cellular homologue of a viral oncogene v-Akt [3] and has therefore also been termed c-Akt. The current interest in PKB stems firstly from the discovery that it is activated rapidly in response to insulin and growth factors and that the activation is prevented by inhibitors of phosphoinositide (PI) 3-kinase [4-6]; secondly, from the finding that PKB isoforms are overexpressed in a significant percentage of ovarian, pancreatic [7, 8] and breast cancer cells [2].
PKB appears to mediate the insulin-induced inhibition of glycogen synthase kinase-3 (GSK3) in L6 myotubes which is thought to underlie, at least in part, the insulin-induced dephosphorylation and activation of glycogen synthase [9] and protein synthesis initiation factor eIF2 [10] that contribute to the stimulation of glycogen and protein synthesis by insulin. However PKB is likely to have other physiological substrates and, in transfection based experiments, has been shown to activate p70 S6 kinase [5], to stimulate the translocation of the glucose transporter GLUT4 to the plasma membrane and enhance glucose uptake into 3T3-L1 adipocytes [11], and to mediate the IGF1-induced survival of neurones [12] and fibroblasts [13] against apoptosis.
A critical question concerns the mechanism by which PI 3-kinase triggers the activation of PKB. The activation of PKB is accompanied by its phosphorylation [5,14] and we recently showed that activation by insulin or IGF1 resulted from its phosphorylation at Thr-308 and Ser-473 [15].
Moreover, the insulin or IGF1 induced phosphorylation of both residues was abolished by wortmannin, an inhibitor of PI 3-kinase [15]. We believe that the protein kinases which phosphorylate PKB at Thr-308 and Ser-473 might themselves be activated by phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), the product of the PI 3-kinase reaction. In the work presented here, we demonstrate that this is indeed the case, and we describe the purification and characterisation of a 3-phosphoinositide-dependent protein kinase (PDK1) which activates PKB and we disclose polynucleotides which encode PDK1 and uses for PDK1 and said polynucleotides.
A first aspect of the invention provides a substantially pure 3-phosphoinositide dependent protein kinase that phosphorylates and activates protein kinase Bxcex1.
By xe2x80x9csubstantially purexe2x80x9d we mean that the 3-phosphoinositide-dependent protein kinase is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said 3-phosphoinositide-dependent protein kinase, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said 3-phosphoinositide-dependent protein kinase.
Thus, the invention also includes compositions comprising the 3-phosphoinositide-dependent protein kinase and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.
The invention also includes the substantially pure said 3-phosphoinositide-endent protein kinase when combined with other components ex vivo, said other components not being all of the components found in the cell in which said protein kinase is found.
It is preferred that the substantially pure 3-phosphoinositide-dependent protein kinase is a substantially pure phosphatidyl-3,4-5-trisphosphate-dependent protein kinase or a substantially pure phosphatidyl-3,4-bisphosphate-dependent protein kinase.
By xe2x80x9cphosphorylates protein kinase Bxcex1xe2x80x9d we include the meaning that the 3-phosphoinositide-dependent protein kinase is able to transfer a phosphate group from ATP to an acceptor group of protein kinase Bxcex1. Preferably, the acceptor group is Thr-308.
By xe2x80x9cprotein kinase Bxcex1xe2x80x9d we include any protein kinase Bxcex1 or any suitable derivative or fragment thereof or fusion of protein kinase Bxcex1 or derivative, or fragment thereof. For example, it is particularly preferred that the protein kinase Bxcex1 is a fusion between glutathione-S-transferase and protein kinase Bxcex1 as described in Example 1 (GST-PKBxcex1; see also reference 27).
It is preferred that the PKBxcex1 is a human PKBxcex1. It should be appreciated that the said 3-phosphoinositide-dependent protein kinase from one species or tissue can phosphorylate and activate PKBxcex1 from another species or tissue.
By xe2x80x9cactivates protein kinase Bxcex1xe2x80x9d we include the meaning that upon phosphorylation by the said 3-phosphoinositide-dependent protein kinase the activity of protein kinase Bxcex1 to a given substrate increases by at least ten-fold compared to the protein kinase Bxcex1 which has not been so phosphorylated, preferably by at least 20-fold and more preferably by at least 30-fold. Suitably, the activity of protein kinase Bxcex1 is measured using the synthetic peptide RPRAATF (SEQ ID NO: 9).
By xe2x80x9c3-phosphoinositide-dependent protein kinasexe2x80x9d we include the meaning that the protein kinase is substantially inactive at activating PKBxcex1 in the absence of a suitable 3-phosphoinositide (or a compound that mimics the effect of a 3-phosphoinositide). In particular, the said protein kinase has at least ten-fold increased activity towards protein kinase Bxcex1 in the presence of a 3-phosphoinositide compared to the activity in the absence of said 3-phosphoinositide, preferably at least 100-fold, more preferably at least 1000-fold, and still more preferably at least 10,000-fold.
It will be appreciated that the 3-phosphoinositide-dependent protein kinase may be activated by mimics of 3-phosphoinositide as described in more detail below.
Preferably, the activation of PKBxcex1 by the 3-phosphoinositide-dependent protein kinase is substantially accelerated by the D-enantiomer of sn-1-stearoyl-2-arachidonyl phosphatidylinositol 3,4,5-trisphosphate but is not substantially accelerated by the L-enantiomer of the said phosphatidylinositol 3,4,5-trisphosphate.
Preferably, the 3-phosphoinositide-dependent protein kinase is substantially activated by the D-enantiomer of sn-1,2-dipalmitoyl phosphatidylinositol 3,4,5-trisphosphate or sn-1,2-dipalmitoyl phosphatidylinositol 3,4-bisphosphate but is not substantially activated by the L-enantiomers of the said phosphatidylinositol phosphates.
Preferably, the 3-phosphinositide-dependent protein kinase is not substantially activated by phosphatidylinositol 3,5-bisphosphate or phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3-phosphate or inositol 1,3,4,5-tetrakisphosphate.
Thus, particularly with reference to FIG. 6 and the rabbit skeletal muscle PDK1 of Example 1, the following 3-phosphoinositides have been found to activate the said 3-phosphoinositide-dependent kinase (in order of level of activation; most effective first):
1. Lipid 5: Racemic sn-1,2-dilinoleoyl PtdIns(3,4,5)P3.
2. (Equal) Lipid 2: D-enantiomer of sn-1-stearoyl-2-arachidonyl PtdIns(3,4,5)P3.
2. (Equal) Lipid 3: D-enantiomer of sn-2-arachidonyl-3-stearoyl PtdIns(3,4,5)P3.
4. (Equal) Lipid 6: sn-1,2 di-palmitoyl PtdIns(3,4,5)P3.
4. Equal Lipid 7: sn-1,2 di-palmitoyl PtdIns(3,4)P2.
The following phospholipids cause no significant activation, at least in relation to rabbit skeletal muscle PDK1:
6. Lipid 2: L-enantiomer of sn-1-stearoyl-2-arachidonyl PtdIns(3,4,5)P3.
7. Lipid 4: L-enantiomer of sn-2-arachidonyl-3-stearoyl PtdIns(3,4,5)P3.
8. Lipid 9: PtdIns(4,5)P2.
9. Lipid 8: sn-1,2 di-palmitoyl PtdIns(3,5)P2.
10. Lipid 11: sn-1,2 di-palmitoyl PtdIns-3P.
11. Lipid 10: PtdIns 4P.
12. IP4: Ins(1,3,4,5P4.
We have found that the human PDK1 enzyme has substantially the same lipid preference as described above.
It is preferred if the 3-phosphoinositide-dependent protein kinase is substantially unaffected by wortmannin.
It should be appreciated that the said 3-phosphoinositide-dependent protein kinase that phosphorylate and activates protein kinase Bxcex1 is likely to phosphorylate and activate other forms of protein kinase B such as protein kinase Bxcex2 and protein kinase Bxcex3. We have shown that PDK1 phosphorylates and activates not only PKBxcex1 but also PKBxcex2 and PKBxcex3. A protein kinase Bxcex2 is described in reference 7. A protein kinase Bxcex3 is described in Konishi et al (1995) Biochem. Biophys. Res. Comm. 216, 526-534.
The 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 may be isolated from any convenient tissue and from any mammal as described below. It is believed that isoforms of the enzyme exist in different tissues within the same mammal and that the invention encompasses said isoforms and said 3-phosphoinositide-dependent protein kinases from any mammal. It is preferred that the said 3-phosphoinositide-dependent protein kinase is the human said enzyme. It is also preferred if it is the rabbit said enzyme.
It is preferred if the said 3-phosphoinositide-dependent protein kinase is about 67 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
A particularly preferred embodiment is a substantially pure 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 the 3-phosphoinositide-dependent protein kinase comprising the amino acid sequences ANSFVGTAQYVSPELL (SEQ ID NO: 3) or AGNEYLIFQK (SEQ ID NO: 4) or LDHPFFVK (SEQ ID NO: 5) or two or more of these sequences or amino sequences with from 1 to 4 conservative substitutions thereof. What is meant by xe2x80x9cconservative substitutionsxe2x80x9d is described below.
A particular preferred embodiment is a polypeptide which comprises the amino acid sequence (SEQ ID NO: 1).
or a variant, fragment, derivative or fusion thereof. The amino acid sequence is that of human PDK1 as determined from the nucleotide sequence of cDNAs encoding human PDK1.
The deduced amino acid sequence is also given in FIG. 10.
As is discussed in more detail in the Examples, at least in relation to human PDK1, experiments in which the pleckstrin homology (PH) domains (which have been found to be present in human PDK1 and which are known to be present in PKBxcex1 and are believed to be involved in binding phospholipids) of either PDK1 or PKBxcex1 were deleted indicated that the binding of PtdIns(3,4,5)P3 or PtdIns(3,4)P2 to PKBxcex1 is required for phosphorylation and activation by PDK1. A GST-PKBxcex1 mutant lacking the PH domain possesses a 3-fold higher activity than that of full length wild-type GST-PKBxcex1 and was activated and phosphorylated by PDK1 in a PtdIns(3,4,5)P3 independent manner; however, the rate of activation was reduced about 20-fold compared to wild-type GST-PKBxcex1. A mutant PDK1 lacking the putative C-terminal PH domain, expressed as a GST-fusion protein, is still able to activate GST-PKBxcex1 in a PtdIns(3,4,5)P3-dependent manner, but the rate of activation was reduced about 30-fold compared to full length GST-PDK1. The effect of PtdIns(3,4,5)P3/PtdIns(3,4)P2 in the activation of PKBxcex1 by PDK1 is, therefore, at least partly substrate directed. However, the drastic reduction in the rate of activation of PKB by PDK1 when the PH domain of PDK1 is deleted indicates the importance of the PH domain of PDK1 and hence the importance of PtdIns(3,4,5)P3/PtdIns(3,4)P2 in the activation of PKB by PDK1. Thus, the substantially pure 3-phosphoinositide dependent protein lipase that phosphorylates and activates protein kinase Bxcex1 may be 3-phosphoinositide dependent in the sense that the intact enzyme (for example intact PDK1) requires the presence of a 3-phosphoinositide in order to phosphorylate an intact PKBxcex1 in vivo. The protein kinase of the invention phosphorylates and activates PKBxcex1 in a 3-phosphoinositide-dependent manner.
PDK1 has been shown by us to phosphorylate p70 S6 kinase in the absence of InsPtd(3,4,5)P3. PDK1 has been shown to bind PtdIns(3,4,5)P3 directly.
A second aspect of the invention provides a recombinant polynucleotide encoding a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 provided that the recombinant polynucleotide is not the DNA corresponding to IMAGE clone 526583 or IMAGE clone 626511. The DNA sequence of at least part of the inserts of these clones are given in GenBank Accession No AA121994 and AA186323, respectively. Preferences for the said 3-phosphoinositide-dependent protein kinase are the same as in the first aspect of the invention. The invention also encompasses polynucleotides which encode variants, fragments, derivatives or fusions of said 3-phosphoinositide-dependent protein kinase or fusions of the said variants, fragments or derivatives. The EST AA121994 was derived from pancreas tissue and the EST AA186323 was derived from HeLa cells.
A particular preferred embodiment of the invention is a polynucleotide comprising the nucleotide sequence (SEQ ID NO: 2).
or variants or variations thereof. The given nucleotide sequence is that containing a coding sequence which encodes human PDK1.
The cDNA sequence is also given in FIG. 10.
IMAGE clones 526583 and 626511 are known in the art and are publicly available from HGMP Resource Centre, I.M.A.G.E. Consortium, Hinxton, Cambridge CB1 1SB, UK. The clones are partial length cDNAs inserted into pBluescript SKxe2x88x92. It was not known that these clones were derived from a mRNA encoding a protein kinase.
Certain other ESTs are publicly available which encode parts of the PDK1 cDNA namely H97903 (melanocyte), AA018098 (retina), AA18097 (retina), AA019394 (retina), AA019393 (retina, N22904 (melanocyte), W94736 (fetal heart), EST 51985 (gall bladder), N31292 (melanocyte), AA188174 (HeLa cells), AA100210 (colon) and R84271 (retina). The polynucleotides corresponding to these ESTs are not claimed per se but they may be useful in carrying out certain parts of the invention. It had not been shown that these clones were derived from a mRNA encoding a protein ase.
The invention also includes a polynucleotide comprising a fragment of the recombinant polynucleotide of the second aspect of the invention. Preferably, the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length. Such polynucleotides are useful as PCR primers.
The polynucleotide or recombinant polynucleotide may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.
A xe2x80x9cvariationxe2x80x9d of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, xe2x80x9cStrategies and Applications of In Vitro Mutagenesis,xe2x80x9d Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, the polynucleotide of the invention includes polynucleotide that shows at least 55 per cent, preferably 60 per cent, and more preferably at least 70 per cent and most preferably at least 90 per cent homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.
DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1xc3x97SSC and 6xc3x97SSC and at temperatures of between 55xc2x0 C. and 70xc2x0 C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By xe2x80x9chigh stringencyxe2x80x9d we mean 2xc3x97SSC and 65xc2x0 C. 1xc3x97SSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.
xe2x80x9cVariationsxe2x80x9d of the polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 80% and preferably at least 90 or 95%) with equivalent stretches of the polynucleotide of the invention even though the overall homology between the two polynucleotides may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.
By xe2x80x9cvariantsxe2x80x9d of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the activity of the said 3-phosphoinositide-dependent protein kinase.
By xe2x80x9cconservative substitutionsxe2x80x9d is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
Such variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art.
Preferably, the variant or variation of the polynucleotide encodes a 3-phosphoinositide-dependent protein kinase that has at least 30%, preferably at least 50% and more preferably at least 70% of the activity towards protein kinase Bxcex1 of a natural said 3-phosphoinositide-dependent protein kinase, under the same assay conditions.
By xe2x80x9cfragment of said 3-phosphoinositide-dependent protein kinasexe2x80x9d we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding assay.
By xe2x80x9cfusion of said 3-phosphoinositide-dependent protein kinasexe2x80x9d we include said protein kinase fused to any other polypeptide. For example, the said protein kinase may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said protein kinase. Fusions to any variant, fragment or derivative of said protein kinase are also included in the scope of the invention.
A further aspect of the invention provides a replicable vector comprising a recombinant polynucleotide encoding a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1, or a variant, fragment, derivative or fusion of said protein kinase or a fusion of said variant, fragment or derivative.
A variety of methods have been developed to operably link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3-single-stranded termini with their 3xe2x80x2-5xe2x80x2-exonucleolytic activities, and fill in recessed 3xe2x80x2-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
In this method the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention. Thus, the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in U.S. Pat. No. 4,440,859 issued Apr. 3, 1984 to Rutter et al, U.S. Pat. No. 4,530,901 issued Jul. 23, 1985 to Weissman, U.S. Pat. No. 4,582,800 issued Apr. 15, 1986 to Crowl, U.S. Pat. No. 4,677,063 issued Jun. 30, 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued Jul. 7, 1987 to Goeddel, U.S. Pat. No. 4,704,362 issued Nov. 3, 1987 to Itakura et al, U.S. Pat. No. 4,710,463 issued Dec. 1, 1987 to Murray, U.S. Pat. No. 4,757,006 issued Jul. 12, 1988 to Toole, Jr. et al, U.S. Pat. No. 4,766,075 issued Aug. 23, 1988 to Goeddel et al and U.S. Pat. No. 4,810,648 issued Mar. 7, 1989 to Stalker, all of which are incorporated herein by reference.
The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
The vectors include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403406 and pRS413416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413416 are Yeast Centromere plasmids (YCps).
The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293 cells which are human embryonic kidney cells. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA.
Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.
For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5xc3x97PEB using 6250V per cm at 25 xcexcFD.
Methods for transformation of yeast by electroporation are disclosed in Becker and Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.
In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
A further aspect of the invention provides a method of making a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said 3-phosphoinositide-dependent protein kinase, and isolating said protein kinase or a variant, derivative, fragment or fusion thereof of a fusion of a variant, fragment or derivative from said host cell. Methods of cultivating host cells and isolating recombinant proteins are well known in the art.
A still further aspect of the invention provides a method of isolating a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 the method comprising the steps of (a) obtaining tissue from a mammal that contains said 3-phosphoinositide-dependent kinase, (b) obtaining a cell-free extract from said tissue, (c) fractionating said cell-free extract and (d) selecting a fraction from step (c) which is capable of phosphorylating and activating protein kinase Bxcex1 in the presence of a 3-phosphoinositide.
Preferably, the 3-phosphoinositide is any of the preferred 3-phosphoinositides as disclosed in relation to the first aspect of the invention; most preferably it is phosphatidylinositol-3,4,5-trisphosphate or phosphatidylinositol-3,4-bisphosphate. Preferably, further steps are employed. Conveniently, in a step (e) the fraction of step (d) is fractionated further and in a step (f) a fraction is selected from step (e) which is capable of phosphorylating and activating protein kinase Bxcex1 in the presence of a 3-phosphoinositide. Steps (e) and (f) may be repeated until a substantially pure preparation of a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1 is obtained. Suitably, the fractionation steps include any of ion exchange chromatography, polyethylene glycol (PEG) precipitation, heparin chromatography or any other fractionation procedures. Preferably, the method steps are those substantially as described in Example 1.
Conveniently, the tissue is skeletal muscle. The tissue may be from any mammal including humans.
A further aspect of the invention provides a phosphoinositide-dependent protein kinase which can phosphorylate and activate protein kinase Bxcex1, or a variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative obtainable by the methods herein disclosed.
A still further aspect of the invention provides an antibody reactive towards a phosphoinositide-dependent protein kinase of the invention.
Antibodies reactive towards the said phosphoinositide-dependent protein kinase of the invention may be made by methods well known in the art. In particular, the antibodies may be polyclonal or monoclonal.
Suitable monoclonal antibodies which are reactive towards the said protein kinase may be prepared by known techniques, for example those disclosed in xe2x80x9cMonoclonal Antibodies: A manual of techniquesxe2x80x9d, H Zola (CRC Press, 1988) and in xe2x80x9cMonoclonal Hybridoma Antibodies: Techniques and Applicationsxe2x80x9d, SGR Hurrell (CRC Press, 1982).
In a preferred embodiment the antibody is raised using any one of the peptide sequences ANSFVGTAQYVSPELL (SEQ. ID NO: 3) or AGNEYLIFQK (SEQ. ID NO: 4) or LDHPFFVK (SEQ. ID NO: 5). It is preferred if polyclonal antipeptide antibodies are made. Other peptides may be used to make antibodies for example the peptides RQRYQSHPDAAVQ (SEQ. ID NO: 6) and LSPESKQARANS (SEQ. ID NO: 7). Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised, may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged. Such modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism. The peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the peptide to be formed as a loop, with the N-terminal and C-terminal ends joined together, or to add one-or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the peptide is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the peptide of the invention forms a loop.
According to current immunological theories, a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope. The peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin. More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, (SEQ ID NO: 8)beta-galactosidase and the 163-171 peptide of interleukin-1. The latter compound may variously be regarded as a carrier or as an adjuvant or as both. Alternatively, several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking. Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4(N-maleimidomethyl) cyclohexane-1-carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
If the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen""s xe2x80x9cEcosecxe2x80x9d system is an example of such an arrangement.
The peptide of the invention may be linked to other antigens to provide a dual effect.
Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4xe2x80x2-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversedN, N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazolemediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, tinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally vailable from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
A further aspect of the invention provides a method of identifying a compound that modulates the activity of a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1, the method comprising contacting a compound with the said 3-phosphoinositide-dependent protein kinase or a variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether, in the presence of said compound, phosphorylation and activation of a protein kinase B or phosphorylation a suitable substrate of the 3-phosphoinositide-dependent protein kinase is changed compared to the activity of said 3-phosphoinositide-dependent protein kinase or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.
We believe that the said 3-phosphoinositide-dependent protein kinase of the invention is able to phosphorylate not only protein kinase Bxcex1 but also other forms of protein kinase B such as protein kinase Bxcex2 and protein kinase Bxcex3. We have shown that PDK1 is able to phosphorylate PKBxcex1, PKBxcex2 and PKBxcex3. Thus, in this method and the methods described below the protein kinase B can be any protein kinase B such as protein kinase Bxcex1 or protein kinase Bxcex2 or protein kinase Bxcex3. Other substrates of the 3-phosphoinositide-dependent protein kinase may be used in the assay method of the invention, for example p70 S6 kinase. A suitable phospholipid such as PtdIns(3,4,5)P3 is typically present in the screen when a PKB containing a PH domain is used as a substrate but it is not necessary for a 3-phosphoinositide to be present in some circumstances, for example when p70 S6 kinase, or PKB lacking a PH domain, is used as a substrate. Phosphorylation of p70 S6 kinase occurs in the absence of PtdIns(3,4,5)P3.
It will be appreciated that the method can be carried out in vitro using 3-phosphoinositide-dependent protein kinase or a variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative in the presence of a suitable protein kinase B or other suitable substrate. But it will also be appreciated that it may be carried out in vivo, for example using the yeast two-hybrid system to detect compounds which reduce or enhance the interactions between the phosphoinositide-dependent protein kinase and PKB or another suitable substrate.
In one embodiment the compound decreases the activity of the 3-phosphoinositide-dependent protein kinase.
In another embodiment the compound increases the activity of the 3-phosphoinositide-dependent protein kinase. Preferably the compound competes with 3-phosphoinositide. Preferably, the compound substantially reduces activation by phosphatidylinositol-3,4,5-trisphosphate or phosphatidyl-inositol-3,4-bisphosphate. Preferably, the compound substantially enhances activation by phosphatidylinositol-3,4,5-trisphosphate or phosphatidylinositol-3,4-bisphosphate. It will be appreciated that the method may be used to identify compounds which bind to and effect the activity of either the said 3-phosphoinositide-dependent protein kinase or the protein kinase B when protein kinase B is present in the assay.
A still further aspect of the invention provides a method of identifying a compound that mimics the effect of a 3-phosphoinositide on a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates protein kinase Bxcex1, the method comprising determining whether said compound activates a said 3-phosphoinositide-dependent protein kinase or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative so that it can phosphorylate and activate protein kinase Bxcex1 or phosphorylate any other suitable substrate, the activation by said compound being in the absence of a 3-phosphoinositide.
The activity of protein kinase B can be measured using any suitable substrate; for example, the peptide RPRAATF (SEQ. ID NO: 9) is a preferred substrate but, for example, myelin basic protein and certain histones are also substrates of PKB.
Preferably, the 3-phosphoinositide is phosphatidylinositol-3,4,5-trisphosphate or phosphatidylinositol-3,4-bisphosphate.
We believe that PtdIns(3,4,5)P3 or a suitable phosphoinositide interacts with the PH domain of a PKB in order for PDK1 to phosphorylate Thr-308 of PKBxcex1 and activate this protein kinase. Our data suggests that a PKB molecule lacking the PH domain is activated and phosphorylated independently of PtdIns(3,4,5)P3. There are several mechanisms by which a drug (which interferes with the interaction between the kinase of the invention, for example, PDK1 and PKB) may act. In one mechanism it may bind to PKB and prevent PKB from becoming activated by the kinase of the invention, for example PDK1. In the other mechanism, the drug may bind to the kinase of the invention (such as PDK1) and inhibit PDK1 activity. In a further mechanism the drug may bind to PDK1 and prevent it activating PKB. It is preferred if the screening assays of the invention are carried out using both a full-length PKB molecule (for example a GST-PKB fusion) or a molecule which, although not full length, retains a PH domain which is substantially capable of binding a suitable phospholipid such as PtdIns(3,4,5)P3, and a PKB in which the PH domain has been modified so that the PKB is substantially incapable of binding a suitable phospholipid such as PtdIns(3,4,5)P3. Suitable modifications include deletion of all or part of the PH domain or mutations in the PH domain which substantially prevent binding of the suitable phospholipid. Compounds which prevent PtdIns(3,4,5)P3 from interacting with PKB are particularly selected, which can be detected in the screen.
Alternatively, it may be useful to use PKB lacking a functional PH domain in a screening assay of the invention, in this case no 3-phosphoinositide need be used in order for PDK1 to activate PKB.
A further aspect of the invention provides a compound identifiable by the screening methods disclosed herein.
The methods effectively are screening assays for compounds which modulate the said 3-phosphoinositide-dependent protein kinase or its interactions with a 3-phosphoinositide or with a protein kinase B.
Thus, the screening methods of the invention include methods for identifying compounds which compete with the 3-phosphoinositide (in particularphosphatidylinositol-3 ,4,5-trisphosphate orphosphatidylinositol-3,4-bisphosphate) and which lead to inactivation or activation of the said 3-phosphoinositide-dependent protein kinase or other modulation of the enzyme activity; they also include screening methods for substances which bind to the said 3-phosphoinositide-dependent protein kinase and which reduce or enhance the activation of a protein kinase B or another suitable substrate (for example, they may bind to said 3-phosphoinositide-dependent protein kinase and prevent it interacting with a protein kinase B); and they also include screening methods for substances which bind to a protein kinase B and which reduce or enhance its interaction with said 3-phosphoinositide-dependent protein kinase. The screening methods of the invention also include methods for identifying compounds which block the catalytic sites of PKB and the protein kinase of the invention (for example, PDK1). Methods for carrying out this type of screening assay are well known in the art.
It will be appreciated that in those assays where a said 3-phosphoinositide-dependent protein kinase is required and the phosphorylation of a particular substrate requires the presence of a 3-phosphoinositide, a 3-phosphoinositide is present. Any suitable 3-phosphoinositide is useful but it is preferred if the 3-phosphoinositide is phosphatidylinositol-3,4,5-trisphosphate or phosphatidylinositol-3,4-bisphosphate. However, as is clear from the foregoing, some assay systems do not require the presence of a 3-phosphoinositide such as PtdIns(3,4,5)P3.
The compounds identified in the methods may themselves be useful as drug or they may represent lead compounds for the design and synthesis of more efficacious compounds which modulate the activity of the said 3-phosphoinositide-dependent protein kinase or its interactions with a 3-phosphoinositide or with a protein kinase B.
Thus, a further aspect of the invention provides a method of modulating in a cell the activity of the said 3-phosphoinositide-dependent protein kinase or its interactions with a 3-phosphoinositide or with a protein kinase B, the method comprising introducing into the cell a compound identifiable in the screening assay described above. Preferably, the cell is in a human patient. Compounds, identifiable in the screening method, which mimic the effect of a 3-phosphoinositide, preferably phosphoinositol-3,4,5-trisphosphate or phosphoinositol-3,4-bisphosphate, are believed to be useful in treating diabetes. Compounds identifiable in the screening methods of the invention that inhibit PKB, PDK1 or the activation of PKB by PDK1 are believed to be useful in treating cancer. PKB is the cellular homologue of v-akt which is involved in leukaemias. Two isoforms of PKB are overexpressed in ovarian, pancratic and breast cancers. It is believed that PKB mediates protection of cells to apoptosis mediated, for example, by IGF-1. Overexpression of PKB may allow cancer cells to proliferate by stopping apoptosis.
It will be appreciated that certain compounds found in the screening methods may be able to enhance cell proliferation in a beneficial way and may be useful, for example in the regeneration of nerves or in wound healing.
Further aspects of the invention provide the use of said 3-phosphoinositide-dependent protein kinase or variants, fragments, derivatives or fusions thereof or fusions of said variants, fragments or derivatives in a screening assay for compounds which modulate the activity of said protein kinase, in particular its interaction with 3-phosphoinositide or a protein kinase B. The invention also includes the use of the said 3-phosphoinositide-dependent protein kinase, such as PDK1, to phosphorylate and active protein kinase B and it includes a method of activating PKB using a 3-phosphoinositide-dependent protein, such as PDK1.
A still further aspect of the invention provides kits of parts that are useful in carrying out the screening methods. Conveniently, the kit of parts comprises a said 3-phosphoinositide-dependent protein kinase or a suitable variant or fragment or derivative or fusion thereof or a fusion of a variant or fragment or derivative (or a polynucleotide which encodes any of these) and a protein kinase B or a suitable variant or fragment or derivative or fusion thereof or a fusion of a variant or fragment or derivative (or a polynucleotide which encodes any of these). It will be appreciated that, depending on the screening method employed, it may additionally comprise a suitable 3-phosphoinositide or a suitable substrate for the protein kinase B.
Abbreviations: PKB, Protein kinase B; PtdIns(3,4,5)P3, Phosphatidylinositol 3,4,5-tris phosphate; PtdIns(3,4,)P2, Phosphatidylinositol 3,4-bisphosphate; PI 3-kinase, Phosphoinositide 3-kinase; PtdCho, Phosphatidylcholine; PtdSer, Phosphatidylserine; PH, pleckstrin homology.